Plasma-induced protein immobilization on polymeric surfaces

ABSTRACT

The present invention relates to a method of immobilizing proteins on a polymeric matrix by means of plasma activation and an apparatus and process for the use of such material. The protein mixture is applied to the surface of the polymeric matrix with or without the addition of a crosslinking agent. It is then placed into a plasma generator, wherein the functional groups on both the protein and the matrix molecules are activated to form free radicals. Upon returning from their high energy state, the free radicals form covalent bonds between the proteins and between the protein and the polymeric matrix. Using this method, the proteins are nonspecifically immobilized on the surface of the polymeric matrix. The method can be utilized to immobilize proteins on the surfaces of polymeric membranes, polymeric beads, polymeric tubes and polymeric plates. The immobilized protein has high biological activity and stability.

This is a division of application Ser. No. 687,224, filed Apr. 18, 1991,now U.S. Pat. No. 5,171,779, which, in turn, is a division ofapplication Ser. No. 220,570 filed Jul. 18, 1988, now U.S. Pat. No.5,028,657.

BACKGROUND OF THE INVENTION

The subject invention relates to the use of a plasma activation methodto induce protein immobilization on a polymeric matrix. There are manyprotein immobilization methods which are available to immobilize proteinon different kinds of materials, for example, chemical activation,entrapment and crosslinking. These conventional methods suffer from manyshortcomings such as forming products of low stability and low activityand the inability of any one method to work well with a variety ofproteins.

In the processes of the prior art, plasma is used to immobilize proteinson a membrane surface only to induce polymerization of differentmonomers to entrap the protein molecules therein. This has beendisclosed in Japanese Patents Nos. 57-197034, 59-203951, 59-28476,59-216587 and 61-87699.

SUMMARY OF THE INVENTION

The instant invention relates to a plasma activation method which can beused to immobilize a wide range of proteins on the polymeric matrix byusing substantially one and the same technique.

More specifically, a protein mixture is first prepared with or without acrosslinking agent and then applied to a membrane. The treated membraneis placed into the reaction chamber which acts as a plasma generator anda vacuum drier. The plasma is generated in a nitrogen, oxygen or ammoniaenvironment under the desired conditions of pressure and temperature,for a defined period of time, to properly activate the protein andmembrane molecules without damaging the normal configuration of theprotein molecules. The covalent bonds formed among protein molecules andprotein-membrane molecules anchor the protein to the surface of themembrane and stabilize the protein configuration. After the reaction iscompleted, the membrane is removed from the reaction chamber and washedwith a buffer solution to remove the unbound protein. The treatedmembranes may be stored in the buffer solution for later use or useddirectly.

By using the method of the invention, a protein membrane having goodactivity and stability can be produced. It may be used for manybiotechnological and biomedical applications, for example, as a membranereactor in biotransformation process, as an enzyme membrane inbiosensing instruments, and as an antigen-antibody membrane inimmunoassays.

The novel plasma activation method can be used to immobilize the proteinon the polymeric matrix regardless of the form of the latter's surfaces.Accordingly, the present invention may be used to form myriad biosensorsand many types of protein matrices in large or small quantities,including the large scale production of protein membranes. The use ofthe process for immobilizing glucose oxidase, lactate oxidase,1-glutamate decarboxylase, 1-lysine decarboxylase and invertase,mutarotase, and glucose oxidase mixtures for the biosensing instrumentpurpose has already been established.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the plasma deposition system.

FIG. 2 shows the components of the biosensor.

FIG. 3 is a graph showing the stability of a plasma activated glucoseoxidase.

FIGS. 4A and 4B include two graphs showing that the optimum pH of aplasma treated glucose oxidase membrane ranges from 4.5 to 7.5.

FIGS. 5A and 5B include two graphs showing that the optimum bufferconcentration of a plasma treated glucose oxidase membrane ranges from0.1 to 0.8M in a Na-PB buffer.

FIG. 6 is a graph showing the storage stability of a glucose oxidasemembrane in a glycerol buffer.

FIG. 7 is a graph showing the result of the analysis of the glucosestandard.

FIG. 8 is a graph showing the result of the analysis of a fermentationbroth sample.

FIG. 9 is a graph showing that the optimum pH of a plasma treatedlactate oxidase membrane ranges from 6.5 to 8.0.

FIG. 10 is a graph showing that the optimum buffer concentration of aplasma treated lactate oxidase membrane ranges from 0.05 to 0.1M in aKPB buffer.

FIG. 11 is a graph showing the durability of lactate oxidase membrane.

FIG. 12 is a graph showing that the optimum pH of a plasma treated1-glutamate decarboxylase membrane ranges from 2.5 to 6.5.

FIG. 13 is a graph showing that the optimum buffer concentration of aplasma treated 1-glutamate decarboxylase membrane ranges from 0.1 to3.2M in a citric buffer.

FIG. 14 is a graph showing the storage stability of 1-glutamatedecarboxylase membrane at 4° C.

FIGS. 15A and 15B include two graphs showing that the optimum pH of aplasma treated 1-lysine decarboxylase membrane ranges from 2.5 to 6.5.

FIG. 16 is a graph showing that the optimum buffer concentration of theplasma treated 1-lysine decarboxylase membrane ranges from 0.1 to 3.2Min a citric buffer.

FIG. 17 is a graph showing the result of the analysis of the 1-lysinestandard.

FIG. 18 is a graph showing that the optimum pH of a plasma treatedsucrose enzyme membrane ranges from 6.5 to 7.0.

FIG. 19 is a graph showing that-the optimum buffer concentration of aplasma treated sucrose enzyme membrane ranges from 0.1 to 0.3 M in a KPBbuffer.

DETAILED DESCRIPTION OF THE INVENTION

A wide variety of proteins may be immobilized in accordance with theinstant invention. In addition to those set forth above, these include,for example, glucose oxidase, 1-glutamate decarboxylase, 1-lysinedecarboxylase, urease, uricase, insulin monoclonal antibody, phenoloxidase, alkaline phosphatase, invertase and mutarotase.

Polymeric materials which may be used to immobilize the proteins includepolyolefins such as hydrophobic and hydrophilic polyethylene andpolypropylene and cellulose acetate, e.g., cuprophane film. Hydrophilicpolypropylene membranes are most preferred. It will be understood,however, that many plastics, elastomers, and fibers having similarlyreactive surfaces may be treated with the protein under appropriatereaction conditions.

The particular surface configuration of the polymeric material may varywidely, depending on the desired application. Most preferably a membranetype surface is employed, though beads, plates and tubes mayconveniently be used.

In the plasma generator, a variety of gases may be used to form theplasma. These include nitrogen, oxygen, and ammonia. A typical plasmagenerator is the radio frequency (13.56 MHz) generator PE-II PlasmaSystem made by Technics Co.

The temperature during the plasma generation may range from 0° to 40°C., preferably from 0° to 25° C. The pressure may range from 0.1 to 0.4torr, preferably from 0.1 to 0.2 torr. The power applied to the plasmagenerator will generally range from 25 to 300 watts, preferably from 50to 300 watts. The time necessary for activation ranges from 1 to 10minutes, preferably from 3 to 5 minutes.

As noted above, the plasma activation may take place in the presence ofcrosslinking agents such as glutaraldehyde. The preferred agent andoptimum amount used may be readily determined by those skilled in theart.

After the activation, the treated product is rinsed in a buffer solutionto remove unbound protein. A variety of buffer solutions may be employedfor this washing step, as well as for storage. Examples of buffersinclude phosphate buffers, tris buffers and citric buffers. Theconcentration used may be readily determined and is dependent on theprotein employed.

The immobilized protein may be used in biotransformation or biosensinginstruments, as well as for immunoassays. For instance, immobilizedtyrosinase may be used in the bioconversion of tyrosine to 1 -DOPA andinsulin monoclonal antibody can be used to assay the insulin content inclinical samples. Other than these examples, otter applications ofimmobilized protein are well known in biotechnology and proteinengineering.

As a general matter, in carrying out the invention, a protein solutionis first prepared by dissolving the protein in the buffer with orwithout a crosslinking reagent. The polymeric material is cut intoappropriate shapes and dimensions, washed with distilled water or NAOHsolution to remove any contamination, and dried. The protein solution ispipetted onto the pretreated membrane and spread evenly with a spatulato form the samples.

The plasma apparatus (Technics Co.), shown in FIG. 1, is operated bysequentially turning on the gases (1), the main power (3), the vacuumpump (5) and the water circulation pump (6). The control switch isturned to manual (3) and the plasma power turned on. When the vacuumreaches 0.1 torr, plasma forms. The time is set when the plasma is readyfor use.

The samples are loaded on the lower electrode by setting the controlswitch to auto (3) and turning on the vent switch to open the reactionchamber (2). The reaction chamber is then closed and the processinitiated by pushing the start button (4). The process is then carriedout in accordance with the preset conditions. After the process iscompleted, the reaction chamber is vented and the treated membranesamples removed. The treated protein membrane is washed several timeswith an appropriate buffer for at least 12 hours. The membrane preparedis then stored in a storage solution or used directly.

The protein membrane may be analyzed by using a FTIR spectrophotometer,by protein stain or any known protein determination methods. If theprotein is an enzyme, then it can be analyzed by detection of the enzymeactivity. If the protein is an antibody, then it can be measured byEnzyme Immuno Assay (EIA) or Radio Immuno Assay (RIA).

In the present invention, the term "biosensor" means a device fordetermining the amount of a given substance. As shown in FIG. 2, itconsists of transducer (1), analyzer (2), recorder (3), enzyme membrane(4), reaction chamber (5), stirrer (6) and temperature controller (7).When the biosensor employs an enzyme as the receptor it is also calledan "enzyme sensor" and, if the enzyme sensor employs an electrochemicaldevice as the transducer portion, then it is called an "enzymeelectrode".

The following examples illustrate specific embodiments of the invention.

EXAMPLE 1: GLUCOSE ENZYME ELECTRODE

A hydrophilic polypropylene membrane is formed into a ring having an 8mm inside diameter ("ID"). The shaped membrane is washed with 0.1Msodium chloride and distilled water, dried and stored in a dessicatorfor later use.

Twenty mg of glucose oxidase (EC 1.1.3.4) Type X (Sigma Co., USA) isdissolved in 1 ml of 0.1M potassium phosphate buffer at pH 5.5. Twentymg of bovine serum albumin are dissolved in 1 ml of the same buffer. Tocomplete enzyme solution preparation, the two solutions are mixedtogether.

On each membrane 5 microliter of enzyme solution is applied to form thesample. The sample is placed in a nitrogen plasma reaction chamber setto 0.1 torr, 125 watts, 4 minutes and 10° C. After treatment, the enzymemembrane is washed several times with 0.1M potassium phosphate buffer atpH 5.5 for 12 hours to remove the unbound proteins.

A kit containing the following components is then prepared as describedbelow:

A) Buffer solution: Dissolve K₂ HPO₄ and KH₂ PO₄ in distilled water tomake 0.1M potassium phosphate buffer, pH 5.5.

B) Glucose standard: Dissolve 0.64 g glucose in 100 ml distilled waterthen serially dilute to make 640, 320, 160, 80 and 40 mg/dl glucosestandard.

C) Serum sample: From the hospital clinical laboratory.

D) Fermentation broth sample: Withdrawn from fermenter and diluted withbuffer in 1:1 ratio to equilibrate the pH difference caused byfermentation process.

To measure the glucose, the instrument shown in FIG. 2 is used. Thetransducer (1) is a Clark's oxygen electrode and the analyzer (2) is anoxygen meter. The recorder (3) is Linseis 6512 strip chart recorder. Thetemperature is set to 30° C. (7).

The glucose oxidase membrane is held on the electrode surface with acuprophane membrane and an O-ring. One-half ml of 1.6 g/dl glucose isadded to adjust the zero oxygen tension. The chamber (5) is washed andrefilled with 4.9 ml buffer to adjust span. When equilibrium isachieved, 0.1 ml of standard is used to develop the calibration curve.The sample is added and the concentration determined from thecalibration curve.

From FIG. 3, it can be seen that the difference between different enzymemembrane prepared with plasma treatment is very small. The optimum pHrange is from pH 4.5 to 7.5 (FIG. 4). The buffer concentration, rangingfrom 0.1M to 0.8M, has no influence on the glucose oxidase membrane(FIG. 5). The membrane, stored in glycerol buffer at 4° C. for 45 days,retains 50% of its original activity as shown in FIG. 6 and thefollowing table:

                  TABLE                                                           ______________________________________                                        Time (Days)                                                                             1      10      20  25    30    45                                   ______________________________________                                        rt-dry    100    44      22  20      8.9   6.7                                rt-buffer 100    87      52  35    26    22                                   rt-glycerol                                                                             100    82      75  48    38    35                                   4° C.-dry                                                                        100    97      70  28    13    12                                   4° C.-buffer                                                                     100    96      54  49    47    41                                   4° C.-glycerol                                                                   100    97      75  60    59    51                                   ______________________________________                                    

The membrane can perform 2400 glucose standard tests (FIG. 7) and 300tests of the fermentation broth samples (FIG. 8).

The glucose oxidase membrane prepared as described above can be used notonly as an enzyme electrode in the fermentation industry, but also forclinical tests for diabetes and in food processing for sweetness tests.It can also be used for the bioconversion of glucose into glutonic acid.

EXAMPLE 2: LACTIC ACID ENZYME ELECTRODE

A hydrophilic polypropylene membrane is cut into a 5 mm ID -ring. Theshaped membrane is washed with 0.1M sodium chloride and distilled water,dried and stored in a dessicator for later use.

Eight mg of glucose oxidase (lactate oxidase from Pediococcus species)(Sigma Co., USA) is dissolved in 1 ml of 0.1M potassium phosphate bufferat pH 7.0. Forty mg of bovine serum albumin are dissolved in 1 ml of thesame buffer. To complete enzyme solution preparation, the two solutionsare mixed together.

On each membrane 1 microliter of enzyme solution is applied to form thesample. The sample is placed in a nitrogen plasma reaction chamber setto 0.1 torr, 125 watts, 4 minutes and 10° C. After treatment, the enzymemembrane is washed several times with 0.1M potassium phosphate buffer atpH 7.0 for 12 hours to remove the unbound proteins.

A kit containing the following components is then prepared as describedbelow:

A) Buffer solution: Dissolve K₂ HPO₄ and KH₂ PO₄ in distilled water tomake 0.1M potassium phosphate buffer, pH 7.0.

B) Lactic acid standard: Dissolve 0.25 gm glucose in 100 ml distilledwater then serially dilute to make 250, 200, 150, 100 and 50 mg/dltactic acid standard.

C) Serum sample: From the hospital clinical laboratory.

D) Fermentation broth sample: Withdrawn from fermenter and diluted withbuffer in 1:1 ratio to equilibrate the pH difference caused byfermentation process.

To measure the lactic acid, the transducer is an Able's hydrogenperoxide electrode and the analyzer is a hydrogen peroxide meter. Therecorder is Linseis 6512 strip chart recorder. The temperature is set to30° C.

The lactate oxidase membrane is held on the electrode surface with acuprophane membrane and an O-ring. One-tenth ml of 2.5 g/dl tactic acidis added to adjust the zero hydrogen peroxide tension. The chamber (5)is washed and refilled with 4.9 ml buffer to adjust span. Whenequilibrium is achieved, 0.1 ml of standard is used to develop thecalibration curve. The sample is added and the concentration determinedfrom the calibration curve.

It was determined that the optimum pH range is from 6.5 to 8.0 (FIG. 9)and that the buffer concentration range of from 0.05m to 0.1M had noinfluence on the lactate oxidase membrane (FIG. 10). The membrane canperform 200 tests on serum samples, while still retaining 30% of itsoriginal activity (FIG. 11). The membrane, stored in glycerol bufferateat 4° C., has activity for over four months.

The lactate oxidase membrane prepared in this manner can be applied notonly to a tactic acid enzyme electrode for clinical, fermentation andfood industries, but also for tactic acid production.

EXAMPLE 3: GLUTAMIC ACID ENZYME ELECTRODE

A hydrophilic polypropylene membrane is cut into an 11 mm ID ring. Theshaped membrane is washed with 0.1M sodium chloride and distilled water,dried and stored in a dessicator for later use.

Twenty-nine mg of 1-glutamate decarboxylase (EC 4.1.1.15) (Sigma Co.,USA) is dissolved in 1 ml of 0.02M citric buffer at pH 4.5 and 60 mg ofbovine serum albumin are dissolved in 1 ml of the same buffer. Inaddition, GA is dissolved in the same buffer to form 0.5 ml of a 1.56%solution. To complete enzyme solution preparation, the two solutions aremixed together.

On each membrane 5 microliter of enzyme solution is applied to form thesample. The sample is placed in a nitrogen plasma reaction chamber setto 0.1 torr, 125 watts, 4 minutes and 10° C. After treatment, the enzymemembrane is washed several times with 0.02M citric buffer at pH 4.5 for12 hours to remove the unbound proteins.

A kit containing the following components is then prepared as describedbelow:

A) Buffer solution: Dissolve 0.214 gm citric acid and 0.389 gm sodiumcitrate in 100 ml distilled water to make 100 ml 0.02M citric buffer, pH4.5.

B) 1-Glutamate standard: Dissolve 0.32 gm 1-glutamate in 100 mldistilled water then serially dilute to make 1.6, 0.8, 0.4 and 0.2 gm/dl1-glutamate standard.

C) Fermentation broth sample: Withdrawn from fermenter and diluted withbuffer in 1:16 ratio to equilibrate the pH difference caused byfermentation process.

To measure the 1-glutamate, the transducer is an ORION carbon dioxideelectrode and the analyzer is an ORION pH meter. The recorder is Linseis6512 strip chart recorder. The temperature is set to 30° C.

The 1-glutamate decarboxylase membrane is held on the electrode surfacewith a cuprophane membrane and an O-ring. Four and one-half ml of bufferare added. When equilibrium is achieved, 0.5 ml of standard is used todevelop the calibration curve. The sample is added and the concentrationdetermined from the calibration curve.

The data show the optimum pH is 4.5 (FIG. 12) and the optimum bufferconcentration range 0.02M for the 1-glutamate decarboxylase membrane(FIG. 13). The membrane, stored in glycerol buffer at 4° C. for 90 days,retained 50% of its original activity (FIG. 14).

The enzyme electrode prepared by the immobilized glutamate decarboxylbsecan be used to detect the glutamic acid content in food additives and infermentation processes.

EXAMPLE 4: LYSINE ENZYME ELECTRODE

A hydrophilic polypropylene membrane is cut into an 11 mm ID ring. Theshaped membrane is washed with 0.1M sodium chloride and distilled water,dried and stored in a dessicator for later use.

Twenty-nine and one-half mg of 1-lysine decarboxylase (EC 4.1.1.18) TypeVII (Sigma Co., USA) is dissolved in 1 ml of 0.01M citrate buffer at pH4.5. Three and three-quarters mg of bovine serum albumin are dissolvedin 1 ml of the same buffer. In addition, GA is dissolved in the samebuffer to form 0.5 ml of a 1.56% solution. To complete enzyme solutionpreparation, the solutions are mixed together.

On each membrane 5 microliter of enzyme solution is applied to form thesample. The sample is placed in a nitrogen plasma reaction chamber setto 0.1 torr, 125 watts, 4 minutes and 10° C. After treatment, the enzymemembrane is washed several times with 0.01M citric buffer at pH 4.5 for12 hours to remove the unbound proteins.

A kit containing the following components is then prepared as describedbelow:

A) Buffer solution: Dissolve 0.11 gm citric acid and 0.20 gm sodiumcitrate in 100 ml distilled water to make 100 ml 0.01 M citric buffer,pH 4.5.

B) I-Lysine standard: Dissolve 18.25 gm lysine in 100 ml distilled waterthen serially dilute to make 9.13, 4.56, 2.28 and 1.14 mg/dl 1-lysinestandard.

To measure the I-lysine, the instrument shown in FIG. 2 is used. Thetransducer is an ORION carbon dioxide electrode and the analyzer is anORION pH meter. The recorder is Linseis 6512 strip chart recorder. Thetemperature is set to 30° C.

The 1-lysine decarboxylase membrane is held on the electrode surfacewith a cuprophane membrane and an O-ring. Four and nine-tenths ml ofbuffer are added. When equilibrium is achieved, 0.1 ml of standard isused to develop the calibration curve. The sample is added and theconcentration determined from the calibration curve.

It can be seen that the optimum pH is 4.5 (FIG. 15) and the optimumbuffer concentration range 0.01M (FIG. 16) for the 1-lysinedecarboxylase membrane. The analysis of the 1-lysine standard is shownin FIG. 17.

The enzyme electrode prepared by the immobilized lysine decarboxylasecan be used to detect the lysine content in food additives and infermentation processes.

EXAMPLE 5: SUCROSE ENZYME ELECTRODE

A hydrophilic polypropylene membrane is cut into a 5 mm ID ring. Theshaped membrane is washed with 0.1M sodium chloride and distilled water,dried and stored in a dessicator for later use.

Forty mg of glucose oxidase, 100 mg invertase, 10 mg mutarotase, 20 mgof bovine serum albumin are dissolved in 1 ml of 0.1M potassiumphosphate buffer at pH 6.5.

On each membrane 2 microliter of enzyme solution is applied to form thesample. The sample is placed in a nitrogen plasma reaction chamber setto 0.1 torr, 125 watts, 4 minutes and 10° C. After treatment, the enzymemembrane is washed several times with 0.01M potassium phosphate bufferat pH 6.5 for 12 hours to remove the unbound proteins.

A kit containing the following components is then prepared as describedbelow:

A) Buffer solution: Dissolve K₂ HPO₄ and KH₂ PO₄ in distilled water tomake 0.1M potassium phosphate buffer, pH 6.5.

B) Sucrose standard: Dissolve 2 gm sucrose in 100 ml distilled waterthen serially dilute to make 2, 1.6, 1.2, 0.8 and 0.4 g/dl sucrosestandard.

C) Serum sample: From the hospital clinical laboratory.

D) Fermentation broth sample: withdrawn from fermenter and diluted withbuffer in 1:1 ratio to equilibrate the pH difference caused byfermentation process.

To measure the sucrose, the transducer is an Able's hydrogen peroxideelectrode and the analyzer is a hydrogen peroxide meter. The recorder isLinseis 6512 strip chart recorder. The temperature is set to 30° C.

The sucrose enzyme membrane is held on the electrode surface with acuprophane membrane and an O-ring. One-tenth ml of 2 g/dl tactic acid isadded to adjust the zero hydrogen peroxide tension. Four and nine-tenthsml of buffer are added. When equilibrium is achieved, 0.1 ml of standardis used to develop the calibration curve. The sample is added and theconcentration determined from the calibration curve.

The data show that the optimum pH range is from 6.5 to 7.0 (FIG. 18).The buffer concentration range from 0.1M to 0.3M has no influence on thesucrose enzyme membrane (FIG. 19). The membrane, stored in glycerolbuffer at 4° C., has activity for over one month.

What is claimed is:
 1. An apparatus for treating, sensing, assaying, ortransforming a biological substance which comprises (a) a reactionchamber; (b) means for supporting a protein immobilized on the surfaceof a polymer in said reaction chamber; (c) means for introducing saidbiological substance into said reaction chamber so as to contact saidimmobilized protein; said protein immobilized to the surface of thepolymer being prepared by coating said polymeric surface with a solutionof said protein; exposing said coated polymer to an environmentcontaining a gas plasma to form free radicals; and covalently bondingsaid protein to said polymeric surface at free radical sites.
 2. Theapparatus of claim 1 wherein said exposure takes place in the presenceof a crosslinking agent.
 3. The apparatus of claim 1 wherein said plasmais a nitrogen, an oxygen, or an ammonia plasma.
 4. The apparatus ofclaim 1 wherein the exposure temperature is from 0° to 25° C.
 5. Theapparatus of claim 1 wherein the exposure pressure is from 0.1 to 0.2torr.
 6. The apparatus of claim 1 wherein the power generated duringsaid exposure is from 50 to 300 watts.
 7. The apparatus of claim 1wherein the polymer is in the form of a membrane.
 8. The apparatus ofclaim 1 wherein the polymer is a bead.
 9. The apparatus of claim 1wherein the polymer is a plate.
 10. The apparatus of claim 1 wherein thepolymer is a tube.
 11. The apparatus of claim 1 wherein the protein isglucose oxidase, lactate oxidase, 1-glutamate decarboxylase, 1-lysinedecarboxylase and invertase, mutarotase, or glucose oxidase mixtures.